Device and method for isolating cell material out of a tissue medium and/or a liquid

ABSTRACT

An apparatus for isolating cell material from a tissue system and/or a liquid includes (1) a vessel open at the top, into which the cell material to be isolated can be introduced in the tissue system and/or the liquid; and (2) a separating device having a stamp-shaped configuration and including a flat separating disk having a peripheral edge fitting with the inner walls of the vessel in a fluid-tight manner and presenting at least one passage opening which is covered by a filter membrane, and which device can be inserted from the top into the vessel. The isolating disk pressurises the cell material inclusive of the tissue system and/or the liquid and acts thereupon with shearing forces by rotation. The cells and/or cell systems pass through the pores of the filter membrane whereas the residual tissue material is retained.

FIELD OF THE INVENTION

The present invention relates to an apparatus for as well as to a methodof isolating cell material from a tissue system and/or a liquid.

PRIOR ART

Individual cells or cell systems are analysed for the medical diagnosisof clinical conditions and for scientific research applications,particularly in the field of bioengineering. The prerequisite for theanalytical methods to be performed on cells is the isolation ofindividual cells from tissue systems or from liquids, respectively,where the individual cells occur in their natural form. In many cases,cells are sampled for the cell analysis from biological soft tissue suchas from the spleen, the liver, and also from harder tissue such as theskin or hair. As part of the analysis of blood or urine, cells arefrequently isolated from liquids, which are then analysed, for instance,to detect pathogenic agents. Such analyses also cover cells contained inbody secretions, smears or other endogenous liquids, which cells,however, must be isolated from the individual tissue systems or liquidslike in all the other aforementioned cases.

So far, however, standardised cell isolation methods have not becomeknown. Predominantly, the manual separation of individual cells or cellagglomerates from tissue systems and/or liquids has been employed so farso that various, individually developed manual methods are applied forextracting cells from tissue, e. g. the spleen, which are adapted to theskill of the respective analysing operator. In this manner, differentcell-isolating techniques have been devised in the course of time, whichwent then through different developments. Moreover, any helpful hintscannot be derived from the pertinent literature from which generallycommon or even standardised methods of universal cell isolation could bederived.

The production of a single-cell suspension for the analysis of aspecific cell type require a number of operating steps to be performedin succession, which have been performed by hand exclusively to date.

For the analysis of cells occurring in tissue systems first of all apiece of tissue, e. g. from the spleen or the liver, must be severed inthe desired size. The removed tissue sample is usually placed into amedium which is introduced, together with the sample, into a flatlaboratory dish. Subsequently, the tissue sample contained in the mediumis mechanically crushed and comminuted by means of suitable tools sothat the cells to be analysed can be better extracted from the tissue.The cells or cell agglomerates, which are concentrated in the medium andare isolated from the individual crushed tissue particles can now beremoved as a suspension from the medium contained in the laboratorydish. Using a pipette whereof the opening can be easily clogged,however, by extracted minor tissue particles usually performs thisseparation. The pipetting of the cell suspension therefore requires therespective operator to apply a particular manual skill because theisolated cells are present in the medium together with the tissueparticles comminuted by the operation of mechanical isolation.

The cell suspension obtained by pipetting is introduced into a vesselagain where the minor tissue fragments, which are removed together withthe cells, can settle on the bottom of the vessel by sedimentationprocesses due to their greater mass. The afore-described pipettingoperation is repeated again in order to obtain in this manner asuspension enriched with the cells. Depending on the type, size andpercentage of extracted small tissue fragments present in thesuspension, the extracting operation, which is based on sedimentation,must be repeated rather frequently. This operation can also be assistedby the application of centrifuges.

For the dissolution and separation of cell systems present in thesuspension into individual cells, the suspension enriched with cellmaterial is extracted by pipetting several times so that as many cellsas possible will be present in an isolated condition in the suspension.The suspension of individual isolated cells from the tissue system,which is obtained in this manner, is usually transferred into anothervessel into which ammonium chloride is added, for instance, for removingthe red blood cells which are equally present in the suspension.

The afore-described methodology of cell isolation, e.g. of cells from atissue fragment, e. g. of the liver, has gone to show that a greatnumber of isolating steps is required which must be performed by hand insuccession until individual isolated cells can be obtained. With each ofthe individual isolating steps, however, residual waste is producedwhich is difficult to discharge, such as the suspension liquids, thetools required for the isolating process like pipetting tips orisolating apparatuses and also the vessels used.

In an approach' to cut the expenditure entailed by the isolatingoperation many laboratories such as diagnostic PCR laboratories desistfrom the specific production of isolated cell suspensions and ratherdigest the complete tissue system. Even though this involves savings interms of time it results in the disadvantage that inhomogeneoushomogenisation products with a plurality of cell types, tissue fragmentsand frequently inhibiting substances for the subsequent analysis areproduced.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is therefore based on the problem of providing anapparatus as well as a method of isolating cell material from a tissuesystem and/or a liquid in such a way that the number of the manualoperating steps to be performed will be reduced and that the cellisolation operation will be largely standardised so as to be able toobtain a reliable reproducibility of the analysis results linked up withthe cell isolation. The waste resulting from each separate isolationoperation, such as the flat laboratory dish as well as general vesselsand tools, should be moreover reduced. In particular, the contaminationof the operating place, which inevitably occurs with the conventionalmethod, should be largely avoided.

The solution to the problem is solved by the apparatus and method ofisolating cell material from a tissue system and/or a liquid accordingto the present invention.

The invention is based on the idea of providing a sample carrier system,which integrates all the functions for cell isolation from a tissue aswell as for accumulation of cells from a liquid.

In accordance with the invention, the apparatus for isolating cellmaterial from a tissue system and/or a liquid comprises a vessel open atthe top, into which the cell material to be isolated can be introducedin the tissue system and/or in the liquid. Moreover, an isolating devicehaving a die-shaped configuration is provided which comprises a flatisolating disk having a peripheral edge which is fluid-tight and flushwith the inside walls of the vessel and presents at least one passageopening covered by a filter membrane. The isolating device can beinserted into the vessel from the top so that the isolating diskpressurises the cell material inclusive of the tissue system and/or theliquid and subjects it, by rotation, to shearing forces. In this systemthe isolating disk is so designed—and preferably provided with grindingelements on its underside—so that the cell material will be comminutedinside the vessel as the isolating disk rotates.

The application of the inventive apparatus makes it possible thatfollowing the sampling operation, e. g. in a physician's office, thetaken sample as well as reagents assisting the process, if this ispossible, can be transferred into the inventive container for transport.With an appropriate dimensioning of the vessel it is possible, at thesame time, to determine the size of the tissue sample to be taken sothat in this manner markings to be applied appropriately on the vesselwill signal to the physician which tissue volume is required for therespective analysis.

This provision equally satisfies the demand for standardisation in theperformance of such tissue analyses.

When the tissue sample in the closable container has arrived in theanalysis laboratory the appropriate reagents can be introduced into thevessel for the subsequent isolating operation in the laboratory. Theisolating device, which presents a die-shaped configuration and which isinserted into the interior of the vessel from the top, is used for cellisolation. In accordance with the present invention, the isolatingdevice is designed to have an isolating disk, which bears flush againstthe inner wall of the vessel and is adapted to be lowered into thevessel via an actuating shaft in a vertical downward direction. Theisolating disk is so configured that it presents passage openings whichare each additionally coated with a filter membrane. The filter membranepresents membrane pores which have a size corresponding to therespective cells or cell systems to be isolated. As the isolating deviceis lowered inside the vessel in a direction towards the tissue samplepresent in a solution, the solution as well as the cells or cell systemsisolated from the tissue sample may pass through the filter membrane andarrive in the upper region of the vessel. In this way two space regionsare created in the vessel which are separated from each other by theisolating disk. The piece of tissue, which is present in the solution,is below the isolating disk whereas the isolated cells or cell systems,which are present in a suspension, are above the isolating disk.

Grinding elements in the form of pointed or angular projections arepreferably provided On the underside of the isolating disk, which so tospeak tear up the tissue sample as the isolating disk is lowered androtated so that the cells or cell systems present in the tissue samplewill be present in the form of a suspension as completely as possibleand thus can pass through the filter membrane into the upper part of thevessel.

The isolating disk can be lowered into the vessel and rotated in acontrolled manner via the actuating shaft which is both mounted ascentral holding shaft on the isolating disk or configured as hollowcylinder flush with the peripheral edge of the isolating disk, so thatcompressing and shearing forces can be selectively exerted on the tissuesample present in the solution. With the assistance afforded by thegrinding elements provided on the isolating disk it is possible torealise a highly efficient separation of the cells or cell systems to beisolated from the tissue sample.

Due to the spatial separation of the cells and cell systems present insuspended form from the comminuted tissue fragments present under theisolating disk, it is possible to remove, by means of usual pipettes,the cell suspension above the isolating disk from the vessel, withoutrunning the risk of small tissue fragments clogging the pipette opening.Subsequent to the appropriate removal of the cell suspension bypipetting or decanting the residual tissue materials and reagentspresent in the vessel below the isolating disk can be disposed oftogether with the vessel.

The inventive apparatus makes it hence possible to implement a samplecarrier and cell isolation system which is suitable for both receivingthe tissue samples and the transport into an analysis laboratory, aswell as the entire isolating process and the final discharge, despitethe high demands on sterility. With the specification of suitable volumesizes of the containers a better standardisation of tissue analyses canbe achieved so that the analysis results can be obtained with a morereliable reproducibility.

Due to the isolating device of the inventive configuration it is firstlypossible to increase the processing rate of the cell isolation operationsubstantially, and secondly to reduce the amount of manual work forperformance of the cell isolation. With the entire isolating processtaking place in one and the same vessel the risks of mutualcontamination with other samples or persons or objects can be reduced toa minimum.

The actuating shaft which is provided for handling the isolating devicecan be expediently operated both manually or even in an automated form.In addition to the provision that the actuating shaft is detachablyfixed to a closure cap closing the vessel and that in this way asimplified manual handling of the isolating device can be implemented,the actuating shaft may also be provided with a flange for automaticactuation by means of an appropriate handling device such as a robotarm. With the application of automatic movers it is possible to performfully automatic cell isolation, using the inventive sample carriersystem. This aspect, in its turn, approaches the aim of a standardisedcell isolation method with the associated high degree of reproducibilitymore closely.

Also in view of the ever-increasing awareness of environmental problems,the inventive sample carrier system contributes to a minimisation of thewaste products to be disposed of as only a single vessel is used.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described by the example of one embodiment withreference to the drawing wherein:

FIG. 1 is a view of the sample carrier system, and

FIG. 2 shows the sequence of operations in the application of the samplecarrier system.

DETAILED DESCRIPTION OF THE DRAWINGS

The inventive sample carrier system consists of a vessel 1 having apreferably cylindrical configuration, which has an upper opening in away resembling a beaker. An isolating device consisting of an isolatingdisk 2 and an actuating shaft 3 can be introduced through the upperopening of the vessel 1, which in the embodiment illustrated here isclosed by a screw-on lid 4. The actuating shaft 3 is centrally seated onthe isolating disk 2 and is fixedly connected thereto. On the other sideof the actuating shaft 2 the latter can be detachably fixed to thescrew-on lid 4, which closes the vessel in a fluid-tight manner. Thescrew-on lid 4 presents a septum on its upper surface, which is suitablefor piercing through pipetting needles for removal of the cellsuspension.

By means of the actuating shaft 3 it is possible to transmit selectivemomenta on the isolating disk 2 by driving the actuating shaft 3 forrotation about its shaft axis and/or in a vertical direction. Inparticular, an adapter or a flange is mounted on the upper section ofthe actuating shaft 3 for automatic handler devices such as a robot arm(which are not illustrated in FIG. 1).

The isolating disk 2 has passage openings on its annular surface whichare covered by a filter membrane 5. The pore size of the filter membranedepends on the respective original tissue and the cell size; for theanalysis of liver cells it amounts to roughly 100 μm as a rule.

Grinding elements 6 are provided on the underside of the isolating disk2, which, in the figure illustrated here, are formed as pointed edges.As the isolating device is appropriately lowered, the grinding elements6 drill into the tissue fragment (not illustrated in FIG. 1) presentbelow the isolating disk, and comminute the tissue material in such away that the cells and cell agglomerates contained in the tissue canpreferably be extracted therefrom.

The vessel illustrated in FIG. 1 is made of a synthetic material, whichcan be easily sterilised; for better visual observation of the isolatingprocesses taking place inside the vessel the container is made to betransparent to light.

In an alternative to the geometry of the actuating shaft 3 asillustrated in FIG. 1, this shaft may be configured as hollow cylinder,which joins and is flush with the peripheral outer edge of the isolatingdisk 2. With such a configuration of the actuating shaft it is possibleto make use of the entire area of the isolating disk 2 for the isolationprocess, which means that the area, too, which in the illustratedembodiment is covered by the centrally disposed actuating shaft on theisolating disk, produces a filter effect.

In the representation of process sequences according to FIG. 2 anexpedient way of application of the inventive sample carrier system isshown. In the first sequence view tissue fragments G to be analysed aretransferred into the interior of the vessel 1. Additionally, a liquid isintroduced for dissolving the cells or cell systems contained in thetissue material, such as a saline solution and/or an ammonium chloridesolution for removal of red blood cells.

In the second step, the tissue sample transferred into the vessel,inclusive of the solution, is closed by a closure cap in a fluid-tightmanner and is now available for transport into from the analysislaboratory.

If necessary, further reagents may be supplied for improving theisolating reactions. To this end, either the closure cap must be removedfrom the vessel and the further reagents are then introduced into theinterior of the vessel by means of a pipetting device 7 in accordancewith the sequential view of the illustrated embodiment. In analternative, the closure cap is provided with a septum, which may bepierced by means of appropriate pointed cannulae so as to introduce aliquid into the interior of the vessel whilst contamination with germsis avoided.

In the sequence view 4 the inventive isolating device is lowered in thevessel either by hand or in an automated system by means of a robot armwhilst it is caused to perform rotating movements at the same time sothat the tissue fragments present on the bottom of the vessel aresubjected to pressure and are comminuted under the action of shearingforces. With the outside periphery of the isolating disk 2 bearingagainst the inside of the vessel wall in a fluid-tight manner, theliquid present in the vessel as well as the cells or cell agglomeratesextracted from the tissue material enter the upper region of the vesselexclusively through the filter membrane applied on the isolating disk.

In sequence 5 the cell suspension present above the isolating disk isremoved by pipetting. There is, however, actually no risk of tissuefragments clogging the pipette opening because macroscopic tissuefragments cannot pass through the filter membrane.

Finally, by the end of the analysis according to sequence, nothing butthe container together with the isolating device and the tissue materialcontained therein is left as residual waste.

LIST OF REFERENCE NUMERALS

1 vessel 2 isolating disk 3 actuating shaft 4 screw-on lid 5 filtermembrane 6 grinding elements 7 pipetting device G tissue fragments

What is claimed is:
 1. An apparatus for isolating cell material from atleast one of a tissue system or a liquid, comprising: a vessel open atthe top into which cell material in the tissue system or in the liquidcan be introduced; and an isolating device comprising: an actuatingshaft; and a flat isolating disk having grinding elements on anunderside of the disk facing the bottom of said vessel; having aperipheral edge flush with inner walls of the vessel in a fluid-tightmanner; and having at least one passage opening covered by a filtermembrane, and which can be inserted into said vessel from the top;wherein said grinding elements introduce shearing forces into the cellmaterial as said isolating disk rotates.
 2. An apparatus according toclaim 1, further comprising a closure cap that closes said vessel in afluid-tight manner.
 3. An apparatus according to claim 1, wherein saidisolating disk pressurises said cell material.
 4. An apparatus accordingto claim 1, wherein said isolating device comprises an actuating shaftcentrally disposed on said isolating disk.
 5. An apparatus according toclaim 1, wherein said isolating device comprises an actuating shafthaving the shape of a hollow cylinder which is flush with the peripheraledge.
 6. An apparatus according to claim 5, wherein said isolatingdevice is adapted to be driven via said actuating shaft for at least oneof rotational or vertical movement.
 7. An apparatus according to claim5, wherein an upper end of said actuating shaft is adapted to bedetachably fastened or fixedly mounted on said closure cap.
 8. Anapparatus according to claim 5, wherein said actuating shaft comprises aflange for automatic or manual actuation by a handling device.
 9. Anapparatus according to claim 2, wherein said closure cap is a screw-oncap.
 10. An apparatus according to claim 2, wherein said closure capcomprises a septum for piercing by means of cannulae.
 11. An apparatusaccording to claim 1, wherein said vessel is made of a syntheticmaterial.
 12. An apparatus according to claim 1, wherein said filtermembrane comprises pores that ensure passage of individual cells or cellsystems through the membrane and that retain remaining tissue material.13. An apparatus according to claim 12, wherein said pores have a sizeof approximately 100 μm.
 14. An apparatus according to claim 1, whereinsaid flat isolating disk has a plurality of openings that are covered bythe filter membrane.
 15. An apparatus according to claim 1, wherein saidgrinding elements are pointed edges.
 16. An apparatus for isolating cellmaterial from at least one of a tissue system or a liquid, consistingof: a vessel open at the top into which cell material in the tissuesystem or in the liquid can be introduced; and an isolating devicecomprising: an actuating shaft and a flat isolating disk having grindingelements and having a peripheral edge flush with inner walls of thevessel in a fluid-tight manner and having at least one passage openingcovered by a filter membrane, and which can be inserted into said vesselfrom the top; wherein said grinding elements introduce shearing forcesinto the cell material as said isolating disk rotates.